Biosensor structures for improved point of care testing and methods of manufacture thereof

ABSTRACT

The present invention relates to analytical testing devices and methods for fabricating electrochemical creatinine biosensors, and in particular using point of care electrochemical biosensors for testing for creatinine in samples. For example, the present invention may be directed to a biosensor having an electrode, a first printed layer formed on the electrode and having a first matrix that includes creatinine amidohydrolase (CNH), creatine amidinohydrolase (CRH), and sarcosine oxidase (SOX), and second printed layer formed over the first printed layer and having a second matrix that includes CRH, SOX, and catalase.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/206,636 filed on Mar. 12, 2014, which claims priority toU.S. Provisional Application No. 61/790,078 filed on Mar. 15, 2013, theentireties of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to analytical testing devices comprisingelectrochemical biosensors and methods for fabricating theelectrochemical biosensors. Specifically, the present invention relatesto analytical testing devices comprising electrochemical creatininebiosensors and methods for fabricating the electrochemical creatininebiosensors, and in particular using point of care electrochemicalbiosensors for testing for creatinine in samples.

BACKGROUND OF THE INVENTION

A biosensor is a device for measuring the concentration of an analyte ina biological sample. A typical biosensor comprises a sensitivebiological recognition element able to interact specifically with atarget analyte, and a transducer or detector element that is able totransform the recognition event of the analyte with the biologicalelement into a measurable signal. In contrast with conventionalbioassays, biosensors allow the detection of molecular interactions asthey take place, without requiring auxiliary procedures, making themhighly attractive for biotechnological applications.

Among the various types of biosensors, electrochemical biosensors aretypically based on enzymatic catalysis of a reaction that produces orconsumes electrons. The biosensor substrate usually contains threeelectrodes: a reference electrode, a working electrode and a counterelectrode. The target analyte is typically involved in a reaction thattakes place on the working electrode surface, and the reaction may causeeither electron transfer across a double layer (producing a current) orcan contribute to a double layer potential (producing a voltage).

One such target analyte typically detected using electrochemicalbiosensors is creatinine. Creatinine is the end metabolite within thehuman body when creatine becomes creatine phosphate and is used as anenergy source for muscle contraction. The creatinine produced isfiltered by the kidney glomeruli and then excreted into the urinewithout reabsorption. The determination of creatinine in body fluids isuseful for diagnosing muscle diseases or various kidney diseases such asnephritis and renal insufficiency.

Typically, in order to detect creatinine using an electrochemicalbiosensor the creatinine needs to be reduced to a detectable productsuch as hydrogen peroxide. One such pathway for achieving a detectableproduct includes the enzyme cascade comprising three enzymes (i)creatinine amidohydrolase (CNH) or creatininease, (ii) creatineamidinohydrolase (CRH) or creatinase, and (iii) sarcosine oxidase (SOX).More specifically, the cascade includes using creatinine amidohydrolaseto catalyze the hydrolysis of creatinine to creatine. Thereafter,creatine amidinohydrolase may be used to catalyze the hydrolysis ofcreatine to sarcosine and urea. Finally, sarcosine oxidase may be usedto catalyze the oxidative demethylation of sarcosine to yield glycineand detectable hydrogen peroxide. However, as there is a significantconcentration of endogenous creatine in the blood, the endogenouscreatine significantly effects the determined concentration ofcreatinine because it is an intermediary byproduct of the enzyme cascadeused to detect the creatinine. In other words, the endogenous creatinecauses interference with the detection of creatinine in the samplebecause the endogenous creatine is also reduced to the product hydrogenperoxide via the use of creatine amidinohydrolase and sarcosine oxidase.

Accordingly, to overcome the influence of endogenous creatine on thebiosensor, a screening layer containing the enzymes creatineamidinohydrolase, sarcosine oxidase, and catalase may be used to reducethe concentration of the endogenous creatine in the sample. For example,a typical creatinine biosensor may use a double layer deposited over asensor. One layer may be used to enzymatically convert the creatinine todetectable hydrogen peroxide in an enzyme cascade such as the onedescribed above. The other layer may be used as the screening layer toenzymatically remove the endogenous creatine using an enzyme cascadesuch as the one described above.

Biosensors comprising the aforementioned screening layer typically havea high design complexity and expensive fabrication techniques.Accordingly, the need exists for improved biosensor designs thataddresses endogenous creatine interference and for improved processesfor making such biosensors.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is a biosensor including anelectrode, a first printed layer formed on the electrode comprising afirst matrix that includes CNH, CRH, and SOX, and a second printed layerformed over the first printed layer comprising a second matrix thatincludes CRH, SOX, and catalase.

In some aspects, the biosensor further comprises a third printed layerformed on the first printed layer such that the third printed layer isdisposed between the first printed layer and the second printed layer,the third printed layer comprising a third matrix that includes CRH.

In some embodiments, the biosensor further comprises a fourth printedlayer formed on the second printed layer comprising a fourth matrix thatincludes CRH, SOX, and catalase.

In another embodiment, the present invention is a method ofmanufacturing a biosensor comprising forming an electrode on a wafer,microdispensing a first layer on the electrode comprising a first matrixthat includes CNH, CRH, and SOX, and microdispensing a second layer overthe first layer comprising a second matrix that includes CRH, SOX, andcatalase.

In another embodiment, the present invention is a biosensor comprisingan electrode and a first spun layer formed on the electrode comprising afirst aqueous polymeric matrix having at least one enzyme. The firstspun layer may have a thickness in a range of about 2-5 μm. Thebiosensor may further comprise a second spun layer formed over the firstspun layer comprising a second aqueous polymeric matrix having at leastone enzyme. The second spun layer may have a thickness in a range ofabout 10-20 μm.

In some aspects, the biosensor further comprises a third spun layerformed on the first spun layer such that the third spun layer isdisposed between the first spun layer and the second spun layer. In someembodiments, the third spun layer may comprise a third aqueous polymericmatrix having at least one enzyme, and the third spun layer may have athickness in a range of about 2-5 μm.

In some embodiments, the biosensor further comprises a silane layerformed on the electrode such that the silane layer is disposed betweenthe electrode and the first spun layer.

In yet another embodiment, the present invention is directed to a methodof manufacturing a biosensor comprising forming an electrode on a waferand spin coating a first layer on the electrode comprising a firstaqueous polymeric matrix having at least one enzyme. The first layer mayhave a thickness in a range of about 2-5 μm. The method may furthercomprise spin coating a second layer over the first layer comprising asecond aqueous polymeric matrix having at least one enzyme. The secondlayer may have a thickness in a range of about 10-20 μm.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood in view of the followingnon-limiting figures, in which:

FIG. 1 shows a number of working electrode designs in accordance withsome aspects of the invention;

FIG. 2 shows an electrochemical cell comprising a working electrode andreference/counter electrodes in accordance with some aspects of theinvention;

FIGS. 3 and 4 show processing steps and respective structures inaccordance with some aspects of the present invention;

FIGS. 5 and 6 show creatinine related test data in accordance with someaspects of the invention;

FIGS. 7 and 8 show processing steps and respective structures inaccordance with some aspects of the present invention;

FIGS. 9-12 show creatinine related test data in accordance with someaspects of the invention;

FIGS. 13 and 14 show processing steps and respective structures inaccordance with some aspects of the present invention;

FIGS. 15 and 16 show creatinine related test data in accordance withsome aspects of the invention;

FIGS. 17-20 show processing steps and respective structures inaccordance with some aspects of the present invention;

FIG. 21 shows an isometric view of a disposable sensing device andreader device in accordance with some aspects of the invention

FIG. 22 shows an isometric top view of a cartridge cover in accordancewith some aspects of the invention;

FIG. 23 shows an isometric bottom view of a cartridge cover inaccordance with some aspects of the invention;

FIG. 24 shows a top view of a tape gasket in accordance with someaspects of the invention;

FIG. 25 shows an isometric top view of a cartridge base in accordancewith some aspects of the invention;

FIG. 26 shows a schematic view of the layout of a cartridge inaccordance with some aspects of the invention; and

FIGS. 27A-27E show top, bottom, side, and perspective views of acartridge in a closed position in accordance with some aspects of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention relates to analytical testing devices comprisingelectrochemical biosensors and methods for fabricating theelectrochemical biosensors. Specifically, the present invention relatesto analytical testing devices comprising electrochemical creatininebiosensors and methods for fabricating the electrochemical creatininebiosensors, and in particular using point of care electrochemicalbiosensors for testing for creatinine in samples. In some embodiments,the invention relates to methods of fabricating biosensor structures onchips such that the biosensors operate in a vertical manner and eachbiosensor comprises a number of biolayers fabricated at variousthicknesses for efficiently and effectively attenuating interferents(e.g., creatine) and detecting target analytes (e.g., creatinine).Accordingly, the present invention provides biosensor structures andmethods of manufacturing that advantageously provide for improvedchemical removal of creatine interference.

More specifically, the present invention may be directed to a method ofmicrodispensing (e.g., printing) enzyme-biolayer matrixes onto asubstrate, and the resultant biosensor structures. Advantageously, themicrodispensing processes described herein overcome waste and costproblems associated with traditional spin coating enzyme-biolayermatrixes onto a substrate. For example, traditional spin coatingprocesses waste a large amount of costly enzyme during the film formingprocess, whereas the manufacture of a biosensor using themicrodispensing embodiments of the present invention may providesignificant cost savings over traditional spin coating processes becausethe enzyme-biolayer matrix is drop dispensed using controlled volumes.Advantageously, the drop dispensing wastes very little if anyenzyme-biolayer matrix.

Additionally, the present invention may be directed to alternativemethods of spin coating enzyme-biolayer matrixes onto a substrate, andthe resultant biosensor structures. Advantageously, the spin coatingprocesses described herein overcome layer thickness problems associatedwith traditional spin coating enzyme-biolayer matrixes onto a substrate.For example, traditional spin coating processes only produce layers ofconsistent thickness across a substrate up to about 5 microns, whereasthe manufacture of a biosensor using the spin coating embodiments of thepresent invention provide a means for forming biolayers of up tounexpected ten-fold greater thickness using combinations of spinning anddrying techniques not used previously. Advantageously, this is importantfor the manufacture of biosensors that are consistent across a wafer andfrom wafer to wafer. It is also important for delivering a screeninglayer that eliminates interfering substances, e.g., creatine in acreatinine sensor.

Overview of Biosensor Fabrication

FIGS. 1 and 2 show an enzyme biosensor provided as a unit cellcomprising a catalytic working electrode 10 and a combined reference andcounter electrode 20 in accordance with some aspects of the presentinvention. The working electrode 10 and counter electrode 20 may beconnected by a lead or signal line 25 (e.g., an over-passivated signalline) to respective contact pads 30. The unit cell may be composed of asingle unit cell confined within a rectangular area which is repeated ina square array several hundred times on a single substrate, for example,a silicon wafer, or composed of two separate unit cells that are groupedtogether to make a final device comprising the enzyme biosensor.

FIG. 3 shows a biosensor structure 50 comprising a wafer 55. In someembodiments, the wafer 55 may comprise a bulk silicon or silicon oninsulator (SOI) wafer. More specifically, FIG. 3 shows an exemplarywafer 55 employed as an intermediate structure. The wafer 55 may befabricated using techniques well known to those skilled in the art. Forexample, the wafer 55 may be thermally oxidized and may have a thicknessof about 10 k{acute over (Å)}; however, the invention is not limited tothese dimensions, and the various portions of the wafer 55 may have anydesired thicknesses.

As shown in FIG. 3, the wafer 55 may comprise multiple areas upon whicha working electrode 60 and a reference electrode may be built eithersimultaneously or at separate stages of manufacture. For example, thewafer 55 may comprise a working electrode 60 formed in a first area ofthe wafer and a reference electrode (not shown) formed in a second areaof the wafer. However, the biosensor structure 50 is not limited to onlyone working electrode and one reference electrode. The working electrode60 may be microfabricated as an array of connected microelectrodes, andthe biosensor structure 50 may additionally comprise contact pads andwiring layers (e.g., a lead wire) that may be fabricated eithersimultaneously or at separate stages of manufacture as the workingelectrode and the reference electrode.

More specifically, the working electrode 60 may be formed comprising ametal layer 65 selectively formed over the wafer 55. Particularly, themetal layer 65 may be formed through electroplating by selectiveelectrodeposition on a seed layer or sputtered (e.g., physical vapordeposition PVD) onto an adhesion layer. In accordance, the metal layer65 may be selectively formed over the seed layer or adhesion layer usingconventional materials and biosensor fabrication techniques, such asusing a hard mask or a photoresist. In particular, a photoresist maskmay be formed on the seed layer or adhesion layer such that the metallayer 65 is only electroplated or sputtered to the seed layer oradhesion layer in areas pertaining to electrodes, contact pads, and/orwiring (e.g., forming a working electrode in an area with a diameter inthe range of 10 μm to 1000 μm). For example, the biosensor 50 and metallayer 65 may be patterned using standard contact lithographic techniquesincluding Fusion systems hardening of a OIR906 photoresist. Reactive ionetching (ME) of the metal layer may be performed in a Tegal 6540 usingchlorine.

In some embodiments, the metal layer 65 may be comprised of gold,silver, platinum, and/or iridium, and may have a thickness from200-1,400 {acute over (Å)}, e.g., from 500-900 {acute over (Å)}, with aresistivity from 0.4-5.3 Ω/m. Additionally, the adhesion layer may becomprised of titanium-tungsten (TiW), and may have a thickness from200-1,000 {acute over (Å)}, e.g., from 200-400 {acute over (Å)}, with aresistivity of from 25-32 Ω/m. However, the invention is not limited tothese materials or dimensions, and the metal layer 65 may be comprisedof any desired materials in any desired thicknesses.

In order to attenuate signals from electrochemical interferents andpromote adhesion of subsequent hydrogel films, a permselective layer 70(e.g., a silane layer) may be selectively patterned onto the wafer 55(e.g., over the working electrode). As shown in FIG. 3, thepermselective layer 70 may be formed by initially preparing an alcoholsolution (e.g., 10 g of alcohol solution) of a silane compound,N-(2-aminoethyl)-3-aminopropyltrimethoxysilane or gamma amino silane, bymixing the silane compound (e.g., 2 mL), in water (e.g., 50 mL), andethanol (50 mL) or 2-propanol (50 g). In accordance with some aspects ofthe invention, the alcohol solution may then be deposited (e.g., spincoated) over the wafer 55 to a predetermined thickness. Thepermselective layer 70 may have a thickness of about 80 {acute over(Å)}; however, the invention is not limited to these dimensions, and thevarious portions of the permselective layer 70 may have any desiredthicknesses.

The wafer 55 and the permselective layer 70 may then be heated (e.g.,heated to about 160° C. for about 15 minutes) to dry and set thepermselective layer 70. A photoresist (e.g., a positive photoresist(Shipley, S1813)) may then be deposited (e.g., spin coated) onto thewafer 55 and the permselective layer 70, soft-baked (e.g., baked atabout 100° C. for about 60 seconds), and patterned (e.g., by means ofexposure to ultraviolet light) through a mask. The resist may then bedeveloped (e.g., Shipley, 455) to leave a resist cap over and around theworking electrode 60 and a lead line (not shown). The exposedpermselective layer 70 may then be selectively etched (e.g., plasmaetched using oxygen and carbon tetrafluoride plasma for about 90seconds). The remaining photoresist may then be stripped (e.g., using anacetone bath) followed by a rinsing (e.g., using a 2-propanol bath).

Optionally, the permselective layer 70 may be selectively wet etchedrather than plasma etched. For example, following the patterning of thephoto resist, the wafer 55 may be etched (e.g., etched in a 1/500 folddilution of hydrofluoric acid (10 M) in deionized water) to removeportions of the permselective layer 70 (e.g., polymerizedN-(2-aminoethyl)-3-aminopropyltrimethoxysilane) not protected by thephotoresist. Other protic solvents, such as lower alkanols, may be usedas the solvent for the hydrofluoric acid. Mixtures of protic solventsmay also be used. Typically, the concentration of hydrofluoric acid inthe protic solvent may be in the range of about 0.001 to about 0.01weight percent. The resist cap may then be removed by exposure of thewafer 55 to n-butylacetate followed by ultrasonication (e.g.,ultrasonication for 15 minutes). Thereafter, the wafer 55 may be bakeddry (e.g., baked at about 100° C. for about 60 minutes).

In alternative embodiments, the permselective layer 70 (e.g., the silanelayer) may be fabricated using a “lift-off” process where thephotoresist is first patterned and the permselective layer 70 is coatedover the photoresist. For example, a layer of photoresist (e.g.,positive photoresist (Shipley, S1813)) may be deposited (e.g.,spin-coated) over the wafer 55 and baked to dry (e.g., soft-baked atabout 90° C. for about 30 minutes). The photoresist may then bepatterned as described previously to leave the area over the workingelectrode exposed. An alcohol solution of the permselective layer 70(e.g., 0.5 g/dL solution ofN-(2-aminoethyl)-3-aminopropyltrimethoxysilane in deionized water) maythen be deposited (e.g., spin coated) onto the wafer 55 and baked to dry(e.g., baked at about 90° C. to 250° C. for about 5 to 30 minutes) underan inert atmosphere. Excess permselective layer 70 (e.g., polymerizedsilane) and photoresist may then be removed (e.g., by means ofultrasonication in n-butylacetate for about 15 minutes). After thephotoresist is removed, the wafer 55 may be baked to dry (e.g., baked atabout 160° C. for about 15 minutes).

These fabrication processes should provide an intermediate biosensorstructure 50 comprising a wafer 55 in which the permselective layer 70is localized over the working electrode, as shown in FIG. 3.

In accordance with some aspects of the invention, additionalphotoformable hydrogel layers or biolayers 75 comprising one or moreenzymes may be established on the intermediate biosensor structure 50shown in FIG. 3 to sensitize the working electrode 60 specifically to ananalyte of interest. In various embodiments of the present invention,the biolayers 75 may be formed by either spin coating or bymicrodispensing each layer (as discussed in detail below). Morespecifically, an aqueous enzyme-hydrogel matrix comprising one or moreproteins and a photoformable hydrogel such as styrylpyridiniumpolyvinylalcohol (SBQ-PVA) supplied by Charkit Chemical Corp., Norwalk,Conn. may be utilized for immobilizing enzymes on or near the workingelectrode 60. A sugar or sugar alcohol, such as sucrose, sorbitol, ormannitol, may also be included in the formulation to alter the porosityof the photoformed matrix and to help stabilize the enzymes. Inaccordance with some aspects of the invention, the one or more proteinsmay comprise SOX, CRH, CNH, catalase, lactate oxidase (LOX), glucoseoxidase (GOX), and/or bovine serum albumin (BSA) mixed to obtain theenzymatic activities, water uptake and diffusional properties desired ineach biolayer.

In addition to SBQ-PVA, other photo-crosslinkable materials, such as,epoxies, acrylamides acrylates and other vinyls given that thecombination of protein, crosslinking agent, photoinitiator, and otheradditives may be found to have suitable negative photoresistcharacteristics may be used in accordance with some aspects of thepresent invention. For example, the hydrogel layers may further comprisea matrix comprising PVA, gelatin, acrylamide, polyethyleneglycoldiacrylate, or combinations thereof.

In embodiments comprising spin coating, the biolayers 75 layers may bephotolithographically patterned using ultraviolet light to crosslink thematerial using a mask followed by removal of the non-crosslinkedmaterial such that the biosensor structure 55 is selectively coated. Inembodiments comprising microdispensing, an appropriate quantity of eachcoating may be applied to an area circumscribed by an additionalstructural component (e.g., optional additional structure 80) configuredas a containment device. In accordance with some aspects of theinvention, the biolayers 75 may be formed such that a diameter of eachsuccessive biolayer increases over that of the diameter of the workingelectrode.

In some embodiments, an additional attenuation layer 85 (e.g., a photodefinable layer comprising methyl-silicone polycarbonate (MSP)hydrophobic polymer (Dow WL7154)) may be spin coated or printed over thebiolayers 80 (e.g., the SBQ-PVA/BSA layer) to further attenuate theanalyte from reaching the detection biolayer, while at the same timeallowing oxygen to be in stoichiometric excess. The additionalattenuation layer 85 may be exposed to ultraviolet light through anappropriate mask, and unexposed portions can be developed using adeveloper such as 1,3,5 trimethylbenzene. Vias 90 may be formed throughthe additional attenuation layer 85 using conventional biosensorfabrication techniques, such as etching the additional attenuation layer85 through a mask, which may be a hard mask or a photoresist. The vias90 may be configured such that the sample comes into contact with thephotoformable hydrogel layer 80 allowing the analyte to diffuse to thephotoformable hydrogel layer 80 in a controlled and reproducible manner.

As should be understood, the methods described above may be used in thefabrication of biosensor chips. The resulting biosensor chips can bedistributed by the fabricator in raw wafer form (that is, as a singlewafer that has multiple unpackaged chips), as a bare die, or in apackaged form. In the latter case biosensor chips may be mounted in asingle chip package (such as a plastic carrier, with leads that areaffixed to a circuit board or other higher level carrier) or in amultichip package (such as a ceramic carrier that has either or bothsurface interconnections or buried interconnections). In any case, thebiosensor chips may then be integrated with other chips, discretecircuit elements, and/or other signal processing devices as part ofeither (a) an intermediate product, such as a circuit board, or (b) anend product. The end product can be any product that includes thebiosensor chips such as a cartridge that is designed to deliver fluid tothe sensor and isolate the electronics from the fluid.

Biolayer Fabrication Using Spin Coating

The descriptions of various embodiments of the present invention arepresented hereafter for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations of the various biolayers should be apparentto those of ordinary skill in the art without departing from the scopeand spirit of the described embodiments.

FIG. 4 shows a creatinine biosensor 100 fabricated in accordance withsome aspects of the present invention. For example, the biosensor 100may comprises a base biosensor structure (e.g., wafer 103, workingelectrode 105, and silane layer 107 fabricated as described above withrespect to FIG. 3) and three biolayers (110, 120, and 130) fabricatedusing a spin-coated process as described in further detail below. Morespecifically, biolayer 130 may be configured as two screening biolayers130 a and 130 b each comprising CRH, SOX and Catalase such that creatinemay be selectively screened from a sample. In accordance with someaspects of the invention, the two screening biolayers 130 a and 130 bmay be formed in two separate spin-coating steps. Biolayer 120 may beconfigured as a diffusion biolayer comprising CRH such that the twoscreening biolayers 130 a and 130 b are separated from the creatininedetection biolayer 110. Creatinine detection biolayer 110 may compriseCNH, CRH, and SOX such that the creatinine may be reduced to detectablehydrogen peroxide in the presence of a substrate that is free to diffusethrough the multiple biolayers.

More specifically, biolayer 110 may be formed as an enzyme-hydrogelmatrix including CNH, CRH, SOX, SBQ-PVA, and sucrose in variousconcentrations (e.g., about 1% CNH, about 4% CRH, about 1% SOX, about 4%sucrose, and about 11% SBQ-PVA (wherein all % are in w/w), apredetermined viscosity (e.g., a viscosity in the range of 1.0 to 1.5Pascal-second), a water content of about 80 to 95 weight-percent, and asolid content of about 5 to 20 weight-percent. The biolayer 110 may bespun (e.g., at a speed in a range of 1000 to 2000 rpm) onto the wafer103 and the working electrode 105. For example, biolayer 110 may be spunonto the wafer 103 and working electrode 105 by dispensing about 6 mL ofsolution onto the center of the wafer 103 and spinning at about 1200 rpmfor 4 seconds and then at about 1850 rpm for 15 seconds. In oneembodiment, the spin-coat process should form a biolayer with athickness of about 1-5 μm, preferably 2-5 μm. The biolayer may then besubstantially dried or cured to remove water content of the biolayerbelow a threshold value. For example, the drying of the biolayer mayreduce the water content of the biolayer below a threshold value ofabout 2 weight-percent. After curing, the biosensor structure may beexposed to ultraviolet light (e.g., about 6 mW/cm², for about 10-30seconds) to crosslink the polymer (e.g., SBQ-PVA) through an appropriatemask and developed using water to wash away the non-crosslinked polymer.

Biolayer 120 may be formed as an enzyme-hydrogel matrix including CRH,SBQ-PVA, and sucrose in various concentrations (e.g., about 6% CRH,about 4% sucrose, and about 9% SBQ-PVA (wherein all % are in w/w), apredetermined viscosity (e.g., a viscosity in the range of 1.0 to 1.5Pascal-second), a water content of about 80 to 95 weight-percent, and asolid content of about 5 to 20 weight-percent. The biolayer 120 may bespun onto the wafer 103 and the biolayer 110 as described above. Forexample, biolayer 120 may be spun onto the wafer 103 and the biolayer110 by dispensing about 6 mL of solution onto the center of the wafer103 and spinning at about 1200 rpm for 4 seconds and then at about 1850rpm for 15 seconds. In one embodiment, the spin-coat process should forma biolayer with a thickness of about 1-5 μm, preferably 2-5 μm. Aftercuring as described above, the biosensor structure may be exposed toultraviolet light (e.g., about 6 mW/cm², for about 10-30 seconds)through an appropriate mask. In some embodiments, the biolayer 120 isselectively exposed to UV light to crosslink the polymer (e.g., SBQ-PVA)but not subsequently developed. Instead, biolayer 120 may be left on thewafer in an undeveloped state.

Biolayer 130 a may be formed as an enzyme-hydrogel matrix including CRH,SOX, SBQ-PVA, catalase and sucrose in various concentrations (e.g.,about 5% CRH, about 1% SOX, about 4% sucrose, and about 10% SBQ-PVA(wherein all % are in w/w), a predetermined viscosity (e.g., a viscosityin the range of 1.0 to 1.5 Pascal-second), a water content of about 80to 90 weight-percent, and a solid content of about 5 to 20weight-percent. The biolayer 130 a may be spun onto the wafer 103 andthe biolayer 120 as described above. For example, biolayer 130 a may bespun onto the wafer 103 and the biolayer 120 by dispensing about 6 mL ofsolution onto the center of the wafer 103 and spinning at about 1200 rpmfor 4 seconds and then at about 1850 rpm for 15 seconds. In oneembodiment, due to the fact that biolayer 120 was not developed and leftpresent across all of the wafer 103, a thicker biolayer (e.g., abiolayer with a thickness of about 8-20 μm, preferably 8-10 μm) may beproduced. After curing as described above, the biosensor structure maybe exposed to ultraviolet light (e.g., about 6 mW/cm², for about 10-30seconds) through an appropriate mask and developed to crosslink thepolymer (e.g., SBQ-PVA). Water may then be used to wash away thenon-crosslinked polymer.

Biolayer 130 b may be formed as an enzyme-hydrogel matrix including CRH,SOX, SBQ-PVA, catalase and sucrose in various concentrations (e.g.,about 5% CRH, about 1% SOX, about 4% sucrose, and about 10% SBQ-PVA(wherein all % are in w/w), a predetermined viscosity (e.g., a viscosityin the range of 1.0 to 1.5 Pascal-second), a water content of about 80to 95 weight-percent, and a solid content of about 5 to 20weight-percent. The biolayer 130 b may be spun onto the wafer 103 andthe biolayer 130 a as described above. For example, biolayer 130 b maybe spun onto the wafer 103 and the biolayer 130 a by dispensing about 6mL of solution onto the center of the wafer 103 and spinning at about1200 rpm for 4 seconds and then at about 1850 rpm for 15 seconds. In oneembodiment, the spin-coat process should form a biolayer with athickness of about 1-5 μm. After curing as described above, thebiosensor structure may be exposed to ultraviolet light (e.g., about 6mW/cm², for about 10-30 seconds) to crosslink the polymer (e.g.,SBQ-PVA) through an appropriate mask and developed using water to washaway the non-crosslinked polymer.

As should be understood, biolayer 103 b may be firmly anchored to thewafer 103 and feature 135 serves to round the contour of the biosensorstructure because biolayer 130 b has a larger surface area than theunderlying biolayers. Advantageously, this may allow the biolayers to berobustly held in place in order to withstand any forces associated witha wet saw dicing process, which may be used to singulate the wafer intoindividual biosensors.

In accordance with above-described processes, biolayer 110 may beconfigured as a creatinine detection layer and produces hydrogenperoxide when creatinine is present in the sample. Biolayer 120 may beconfigured to separate the detection layer from the screening layers andprovides additional CRH enzyme for both detection and screeningpurposes. In addition, during the spin coating process biolayer 120assists in increasing the thickness of the third enzyme layer becausebiolayer 120 is not developed and acts as a sponge as discussed indetail below. Biolayer 130 a may be configured to remove creatine fromthe sample before the creatine can reach biolayer 110 where the creatinemay interfere with the detection of creatinine. Biolayer 130 b may alsohelp to eliminate creatine interference and may serve as a cap to anchorthe biolayers to the wafer 103.

A should be understood by those of ordinary skill in the art, theabove-described process provide a modified spin coating process that maybe used to create at least one biolayer thicker (e.g., a layer thicknessof >5-12 μm) than achieved with typical spin coating processes. Themodification comprises leaving an underlying hydrogel layer undevelopedprior to applying a new hydrogel layer. Therefore, the underlyinghydrogel acts as a sponge soaking up water from the new hydrogel layersince the underlying hydrogel has not been developed, which dehydratesor dries the new hydrogel layer as the new hydrogel layer spreads acrossthe biosensor structure. This enhanced drying effect causes a uniformthicker biolayer to be produced. Although the new biolayer may bethicker after exposure, the new biolayer and the underlying biolayer canbe developed thereafter as described above. Advantageously, the spincoating processes described herein overcomes layer thickness limitationsassociated with traditional spin coating enzyme-biolayer matrixes onto asubstrate.

FIG. 5 shows a response, in nanoamps of the creatinine biosensor 100 tosamples with various levels of creatinine (e.g., 0, 1, 2, 6.8, and 13.6mg/dL). During the first 23 seconds, a calibrant solution is in contactwith the creatinine biosensor 100, which is followed by the sample. Thecalibrant response (17-20 seconds) is taken just before the samples areput in front of the creatinine biosensor 100 and the sample signals aretaken at a time when the sample response is at its steepest (27-30seconds). The sensor response may be normalized by dividing the slope ofthe sample response by the corresponding calibrant response of thesensor (DIV). FIG. 6 shows a plot of the DIV response for the variousconcentration of creatinine samples tested. The linearity of theresponse over this range shows a considerable improvement for creatininedetection using the creatinine biosensor 100 over traditional creatininebiosensors.

FIG. 7 shows a creatinine biosensor 200 fabricated using an alternativespin coating process in accordance with some aspects of the presentinvention. For example, the biosensor 200 comprises a base biosensorstructure (e.g., wafer 203, working electrode 205, and silane layer 207fabricated as described above with respect to FIG. 3) and threebiolayers (210, 220, and 230) fabricated using a spin-coated process.More specifically, biolayer 230 may be configured as three screeningbiolayers 230 a, 230 b, and 230 c each comprising CRH and SOX such thatcreatine may be selectively screened from a sample. In accordance withsome aspects of the invention, the three screening biolayers 230 a, 230b, and 230 c may be formed in two or three separate spin-coating steps.Biolayer 220 may be configured as a diffusion biolayer comprising CRHsuch that the three screening biolayers 230 a, 230 b, and 230 c areseparated from the creatinine detection biolayer 210. Creatininedetection biolayer 210 may comprise CNH, CRH, and SOX such thatcreatinine may be reduced to detectable hydrogen peroxide in thepresence of a substrate that is free to diffuse through the multiplebiolayers.

More specifically, biolayer 210 may be formed as an enzyme-hydrogelmatrix as discussed-above with respect to biolayer 110 in FIG. 4.Biolayer 220 may be formed as an enzyme-hydrogel matrix asdiscussed-above with respect to biolayer 120 in FIG. 4 except that thebiolayer 220 is UV exposed to crosslink the polymer (e.g., SBQ-PVA) andsubsequently developed using water to remove the non-crosslinkedpolymer. Biolayer 230 a may be formed as an enzyme-hydrogel matrix asdiscussed-above with respect to biolayer 130 a in FIG. 4 except thatsince biolayer 220 was developed, biolayer 230 a may be formed as athinner layer (e.g., a biolayer with a thickness of about 1-5 μm).Additionally, biolayer 230 a after crosslinking the polymer (e.g.,SBQ-PVA) may not be subsequently developed using water to remove thenon-crosslinked polymer, but may instead be left on the biosensorstructure in an undeveloped state. Biolayer 230 b may be formed as anenzyme-hydrogel matrix as discussed-above with respect to biolayer 130 bin FIG. 4; however, due to the fact that biolayer 230 a was notdeveloped and left present across all of the wafer 203 a thicker layer(e.g., a biolayer with a thickness of about 8-20, preferably 8-10 μm)may be produced. Additionally, biolayer 230 b may not be subsequentlydeveloped using water to remove the non-crosslinked polymer, but mayinstead be left on the biosensor structure in an undeveloped state.Biolayer 230 c may be formed as an enzyme-hydrogel matrix asdiscussed-above with respect to biolayer 130 b in FIG. 4; however, dueto the fact that biolayer 230 b was not developed and left presentacross all of the wafer 203 a thicker layer (e.g., a biolayer with athickness of about 8-20, preferably 8-10 μm) may be produced.Additionally, biolayer 230 c may be exposed to ultraviolet light tocrosslink the polymer (e.g., SBQ-PVA) and subsequently developed usingwater to remove the non-crosslinked polymer. Advantageously, theadditional thick screening biolayer (e.g., biolayer 230 c at about 8-20,preferably 8-10 μm) improves the creatine removal before the creatinereaches the detection biolayer 210.

Biolayer Fabrication Using Microdispensing

The descriptions of various embodiments of the present invention arepresented hereafter for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations of the various biolayers should be apparentto those of ordinary skill in the art without departing from the scopeand spirit of the described embodiments.

FIG. 8 shows a creatinine biosensor 300 fabricated in accordance withsome aspects of the present invention. For example, the biosensor 300may comprise a base biosensor structure (e.g., wafer 303 and workingelectrode 305 fabricated as described above with respect to FIG. 3) andthree biolayers (310, 320, and 330) fabricated using a microdispensingor printing process as described in further detail below. Morespecifically, biolayer 330 may be configured as a screening biolayercomprising CRH, SOX and catalase such that creatine may be selectivelyscreened from a sample. Biolayer 320 may be configured as a diffusionbiolayer comprising CRH such that the screening biolayer is separatedfrom the creatinine detection biolayer 310. Creatinine detectionbiolayer 310 may comprise CNH, CRH, and SOX such that the creatinine maybe reduced to detectable hydrogen peroxide in the presence of asubstrate that is free to diffuse through the multiple biolayers.

More specifically, biolayer 310 may be formed as an enzyme-hydrogelmatrix including CNH, CRH, SOX, SBQ-PVA, and sucrose in variousconcentrations (e.g., about 0.4% CNH, about 1.5% CRH, about 0.2% SOX,about 1% sucrose, and about 3% SBQ-PVA (wherein all % are in w/w) and apredetermined viscosity (e.g., about 0.3 P (0.12 N s m⁻²)). The biolayer310 may be dispensed onto the wafer 303 and the working electrode 305 asdescribed above. For example, the biolayer 310 may be dispensed onto thewafer 303 by use of partial drop transfer of solution onto the workingelectrode 305 to fill a formed additional structure or containment ring340 (e.g., about 1 nL). The biolayer may then be substantially dried orcured to remove water content of the biolayer below a threshold value.For example, the drying of the biolayer may reduce the water content ofthe biolayer below a threshold value of about 2 weight-percent. Afterdrying, the biolayer 310 may be exposed to ultraviolet light (e.g.,about 6 mW/cm², for 10-30 seconds) to crosslink the polymer (e.g.,SBQ-PVA) and developed. In some embodiments, the drying of the biolayer310 may be performed in low relative humidity (e.g., about 5-20% RH).The low relative humidity may result in a cross sectional profile 350 ofthe biolayer 310 that may not be planar (e.g., a substantially concaveshape). Instead, the biolayer 310 has a thick ring 360 around the edge.However, the biolayer 310 is of uniform thickness 370 over the workingelectrode 305 because the extension 380 is configured to absorb thethick ring 360 non-planar profile 350.

Biolayer 320 may be formed as an enzyme-hydrogel matrix including CRH,SBQ-PVA, and sucrose in various concentrations (e.g., about 1.4% CRH,about 1% sucrose, and about 3% SBQ-PVA (wherein all % are in w/w) and apredetermined viscosity (e.g., about 0.4 P (0.13 N s m⁻²)). The biolayer320 may be dispensed onto the wafer 303 and the biolayer 310 asdescribed above. For example, the biolayer 320 may be dispensed onto thewafer 303 by use of partial drop transfer of solution onto the workingelectrode 305 in sufficient volume to completely cover biolayer 310(e.g., about 1 nL). After drying as described above, the biolayer 320may be exposed to ultraviolet light (e.g., about 6 mW/cm², for 10-30seconds) to crosslink the polymer (e.g., SBQ-PVA) and developed. In someembodiments, the underlying biolayer 310 may be configured to absorbwater when the biolayer 320 is dispensed. Thus, biolayer 320 dries morequickly and evenly, and the cross sectional profile of the biolayer 320is more planar than the biolayer 310 even though the same dryingconditions are employed (e.g., about 5-20% RH).

Biolayer 330 may be formed as an enzyme-hydrogel matrix including CRH,SOX, SBQ-PVA, catalase and sucrose in various concentrations (e.g.,about 2.5% CRH, about 0.4% SOX, about 0.4% catalase, about 0.6% sucrose,and about 5.3% SBQ-PVA (wherein all % are in w/w) and a predeterminedviscosity (e.g., about 0.6 P (0.12 N s m⁻²)). The biolayer 330 may bedispensed onto the wafer 303 and the biolayer 320 as described above.For example, the biolayer 330 may be dispensed onto the wafer 303 by useof partial drop transfer of solution onto the biolayer 320 in sufficientvolume to completely cover biolayer 320 (e.g., about 1 nL). Afterallowing this layer to partially dry a second dispense may be performed,which doubles the thickness of the biolayer 330. In some embodiments,underlying biolayer 320 absorbs water when the biolayer 330 isdispensed. Thus, biolayer 330 dries more quickly and evenly, and thecross sectional profile of the biolayer 330 is even more planar than thebiolayer 310 or the biolayer 320 even though the same drying conditionsare employed (e.g., e.g., about 5-20% RH). After drying as describedabove, the biolayer 330 may be exposed to ultraviolet light (e.g., about6 mW/cm², for 10-30 seconds) to crosslink the polymer (e.g., SBQ-PVA)and developed.

In accordance with the above-described processes, biolayer 310 may beconfigured as a creatinine detection layer and produces hydrogenperoxide when creatinine is present in the sample. Biolayer 320 may beconfigured to separate the detection layer from the screening layers andprovides additional CRH enzyme for both detection and screeningpurposes. Biolayer 330 may be configured to remove creatine of thesample before the creatine can reach biolayer 310 where the creatine mayinterfere with the detection of creatinine.

A should be understood by those of ordinary skill in the art, theabove-described microdispensing process may be used to advantageouslycreate at least one biolayer in a controlled and cost efficient fashionas compared to typical spin coating processes. Advantageously, the dropdispensing wastes very little if any enzyme-hydrogel matrix.

FIG. 9 shows a response, in nanoamps of the creatinine biosensor 300 tosamples with various levels of creatinine (e.g., 0, 0.15, 0.6, 1.2mmol/L). During the first 23 seconds, a calibrant solution is in contactwith the creatinine biosensor 300, which is followed by the samples. Thecalibrant response (17-20 seconds) is taken just before the samples areput in front of the creatinine biosensor 300 and the sample signals aretaken when the sample response is most stable (34-39 seconds). Thesensor response may be normalized by dividing the slope of the sampleresponse by the corresponding calibrant response of the sensor (DIV).FIG. 10 shows a plot of the DIV response for the various concentrationof creatinine samples tested. The linearity of the response over thisrange shows a considerable improvement for creatinine detection usingthe creatinine biosensor 300 over traditional creatinine biosensors.

In alternative embodiments, the creatinine biosensor 300 may be modifiedsuch that biolayer 310 is configured as a detection biolayer comprisingCNH such that creatinine is reduced to creatine, and detection biolayer320 comprises CRH, and SOX such that the creatine may be reduced todetectable hydrogen peroxide in the presence of a substrate that is freeto diffuse through the three biolayers. FIG. 11 shows a response, innanoamps of this alternative creatinine biosensor 300 to samples withvarious levels of creatinine (e.g., 0, 0.15, 0.6, 1.2 mmol/L). Duringthe first 23 seconds, a calibrant solution is in contact with thecreatinine biosensor 300, which is followed by the sample. The calibrantresponse (17-20 seconds) is taken just before the samples are put infront of the creatinine biosensor 300 and the sample signals are takenwhen the sample kinetic response is most stable (32-39 seconds). Thesensor response may be normalized by dividing the slope of the sampleresponse by the corresponding calibrant response of the sensor (DIV).FIG. 12 shows a plot of the DIV response for the various concentrationof creatinine samples tested. This plot shows improved chemical removalof creatine interference and a reduced flux of creatine to the detectionlayer. Therefore, a lower detection signal (i.e., reduction of apositive interferent), however the loss of negative slope response forcreatine may be achieved thereby appearing to give less creatinediscrimination when using the slope of the sample signal as theanalytical response.

In alternative embodiments, the creatinine biosensor 300 may be modifiedas shown in FIG. 13 such that the biolayers 310, 320, and 330 areprinted and/or dried in a high humidity environment, e.g., in the rangeof 40 to 98% RH. This process may result in the biolayers that have asubstantially convex shape as shown in FIG. 13.

In alternative embodiments, the creatinine biosensor 300 may be modifiedto comprise only two biolayers as shown in FIG. 14. More specifically,biosensor 300 may be configured to only comprise a detection layercomprising CNH, CRH and SOX (e.g., biolayer 310) in various amounts anda screening layer comprising CRH, SOX and catalase (biolayer 330) invarious amounts. Each biolayer may be produced by printing biolayer 310twice and biolayer 330 twice as described above. FIG. 15 shows aresponse, in nanoamps of this alternative creatinine biosensor 300 tosamples with various levels of creatinine (e.g., 0, 0.15, 0.6, 1.2mmol/L). During the first 23 seconds, a calibrant solution is in contactwith the creatinine biosensor 300, which is followed by the samples. Thecalibrant response (17-20 seconds) is taken just before the samples areput in front of the creatinine biosensor 300 and the sample signals aretaken when the sample response is most stable (30-39 seconds). Thesensor response may be normalized by dividing the slope of the sampleresponse by the corresponding calibrant response of the sensor (DIV).FIG. 16 shows a plot of the DIV response for the various concentrationof creatinine samples tested. This plot shows improved chemical removalof creatine interference and a reduced flux of creatine to the detectionlayer. Therefore, a lower detection signal (i.e., reduction of apositive interferent), however the loss of negative slope response forcreatine may be achieved thereby appearing to give less creatinediscrimination when using the slope of the sample signal as theanalytical response.

Additional Structures for Use With the Aforementioned MicrodispensingProcesses

In alternative or additional embodiments, the creatinine biosensor 300may be modified as shown in FIGS. 17-20 such that the biolayers 310,320, and 330 are printed using additional structures or containmentring(s) to generate a predefined structure. The predefined structure maybe configured to fabricate a lateral flow or vertical flow biosensor.The optional additional structure (e.g., containment ring 340) may beformed prior to dispensing the biolayers and configured to contain thedrops of enzyme-hydrogel matrix. The additional structural component maybe formed of a mask material such as a photoresist material thatincludes various viscosities such that it may be capable of sufficientsidewall definition and does not exhibit chemical incompatibility withthe function of the biosensors structure. For example, in accordancewith some aspects of the invention, a photoresist material may beselected that is an epoxy-based negative photoresist such as SU-8.

For example, as shown in FIGS. 17-20 the lateral flow biosensor 300 maycomprise a base biosensor structure (e.g., wafer 303 and workingelectrode 305 fabricated as described above with respect to FIG. 3) andthree biolayers (310, 320, and 330). The biosensor 300 may be fabricatedusing a printing process to stack the three biolayers one on top of theother inside containment rings 340 and 340′. More specifically,creatinine detection biolayer 310 may comprise CNH, CRH, and SOX suchthat the creatinine may be reduced to detectable hydrogen peroxide inthe presence of a substrate that is free to diffuse through the multiplebiolayers.

As shown in FIG. 17, the biolayer 310 may be formed within thecontainment ring 340 in accordance with similar processing steps usedabove with respect to biolayer 310 in FIGS. 8 and/or 13. Biolayer 320may be configured as a diffusion biolayer comprising CRH such that thescreening biolayer is separated from the creatinine detection biolayer310. As shown in FIG. 18, the biolayer 320 may be formed within thecontainment ring 340 in accordance with similar processing steps usedabove with respect to biolayer 320 in FIGS. 8 and/or 13. Biolayer 330may be configured as a screening biolayer comprising CRH, SOX andcatalase such that creatine may be selectively screened from a sample.As shown in FIG. 19, the biolayer 330 may be formed within thecontainment ring 340′ in accordance with similar processing steps usedabove with respect to biolayer 330 in FIGS. 8 and/or 13. For example,the biolayer 330 may extend into the tabs 382. As shown in FIG. 20, anadditional impermeable layer 384 (e.g., a photo definable layercomprising methyl-silicone polycarbonate (MSP) hydrophobic polymer (DowWL7154)) may be spin-coated or printed over the biolayer 330 in such amanner that the tabs 382 remain uncovered by the impermeable layer 384.Therefore, the tabs 382 may be act as wicks configured to contact thesample and permit the analyte to diffuse through the biolayers in acontrolled and reproducible manner.

Systems Comprising a Biosensor Configured for Target Analyte Detection

Referring to FIG. 21, the system 385 of the present invention maycomprise a self-contained disposable sensing device or cartridge 386 anda reader device or instrument 387. A fluid sample (e.g., whole blood orurine) to be measured is drawn into a sample entry orifice or port 388in the cartridge 386, and the cartridge 386 may be inserted into thereader device 387 through a slotted opening 389. The reader device 387may comprise a processor configured to perform measurements of analyteconcentration within the fluid sample, as discussed herein in furtherdetail. Measurements and determinations performed by the reader may beoutput to a display 390 or other output device, such as a printer ordata management system 391 via a port on the reader 392 to a computerport 393. Transmission can be via Wifi, Bluetooth link, infrared and thelike. Note that where the biosensors 395 are based on electrochemicalprinciples of operation, the biosensors 395 in the cartridge 386 makeelectrical contact with the instrument 387 via an electrical connector397. For example, the connector may be of the design disclosed in U.S.Pat. No. 4,954,087, incorporated herein by reference in its entirety.The instrument 387 may also include a method for automatic fluid flowcompensation in the cartridge 386, as disclosed in U.S. Pat. No.5,821,399, which also is incorporated herein by reference in itsentirety.

In one embodiment, as shown in FIGS. 22-25, a cartridge 400 (e.g., adisposable assay cartridge) may comprise a cover 405 (as shown in FIGS.22 and 23), a base 410 (as shown in FIG. 25), and a thin-film adhesivegasket 415 (as shown in FIG. 24) that is disposed between the base 410and the cover 405. The cartridge 400 may be configured for insertioninto a reader device, and therefore the cartridge 400 may comprise aplurality of mechanical and electrical connections (not shown) for thispurpose. Advantageously, a feature of the cartridge 400 is that once asample is loaded within the cartridge 400, analysis of the sample may becompleted and the cartridge 400 may discarded without an operator orothers contacting the sample.

Referring to FIG. 22, the cover 405 may be made of a rigid material,preferably plastic, and capable of repetitive deformation at flexiblehinge regions 420, 425, and 430 without cracking. The cover 405 maycomprise a lid 435, attached to a main body of the cover 405 by theflexible hinge 425. In operation, after introduction of a sample into asample holding chamber 440 (as shown in FIG. 25) through a sample entryport 445, the lid 435 may be secured over an entrance to the sampleentry port 445, preventing sample leakage. The lid 435 may be held inplace by a hook 450.

The cartridge 400 optionally may also have a closure feature asdescribed in U.S. Pat. No. 7,682,833, which is hereby incorporated byreference in its entirety, for sealing the sample entry port 445 in anair-tight manner. This closure device may be slidable with respect to abody of the cartridge 400 and provides a shearing action that displacesexcess sample located in the region of the sample entry port 445,reliably sealing a portion of the sample in the sample holding chamber440 between the sample entry port 445 and a capillary stop.Specifically, the cartridge 400 may be sealed by slidably moving asealing element over the surface of the cartridge in a manner thatdisplaces excess fluid sample away from the sample entry port 445, sealsa volume of the fluid sample within the internal fluid sample holdingchamber 440, and inhibits fluid sample from prematurely breaking throughthe internal capillary stop.

The cover 405 may further comprise two paddles 455 and 460 that aremoveable relative to the body of the cover 405, and which are attachedto the cover 405 by the flexible hinge regions 420 and 430. The paddle460 may be configured to be operated by a pumping means such that aforce is exerted upon an air bladder comprised of cavity 465 (as shownin FIG. 24) and the gasket 415. Operation of the paddle 460 displacesfluid within conduits of the cartridge 400.

The paddle 455 may be configured to be operated upon by a second pumpingmeans such that a force is exerted upon the gasket 415, which can deformbecause of slits 470 cut therein (as shown in FIG. 24). Deformation ofthe gasket 415 may transmit pressure onto a fluid-containing foil packfilled with a fluid, e.g., approximately 130 μL of analysis/washsolution or fluid, located in cavity 475 (as shown in FIG. 25),rupturing the foil pack upon spike 480, and expelling fluid into conduit485. The conduit 485 may be connected via a short transecting conduit inthe base 410 to a conduit 490 (as shown in FIG. 23). The fluid fills afront of the conduit 485 first pushing fluid into a small opening in thegasket 415 that acts as a capillary stop.

Additional action in the cartridge 400 generated by mechanisms withinthe reading device applied to the cartridge 400 may be used to injectone or more air segments into the fluid at controlled positions withinthe conduit 490. The air segments may be used to wash the biosensorsurface of the sensor array and the surrounding conduit 490 with aminimum amount of fluid. For example, the cover 405 may further comprisea hole covered by a thin pliable film 495. In operation, pressureexerted upon the film 495 may expel one or more air segments into theconduit 490 through a small hole 505 in the gasket 415 (as shown inFIGS. 23 and 24).

Referring to FIG. 23, a lower surface of the cover 405 further comprisesthe conduit 490 and another conduit 510. The conduit 490 includes aconstriction 520 that controls fluid flow by providing resistance to theflow of the fluid. Optional coatings 525 and 530, e.g., dry reagentcoatings, may provide hydrophobic surfaces on the conduit 510, whichtogether with gasket holes 535 and 540 control fluid flow betweenconduits 190 and 510. A recess 545 in the base may provide a pathway forair to enter and/or escape the conduit 440 through hole 550 in thegasket.

Referring to FIG. 24, the thin-film gasket 415 comprises various holesand slits to facilitate transfer of fluid and air between conduitswithin the base 405 and the cover 410, and to allow the gasket 415 todeform under pressure where necessary. Specifically, a hole 555 maypermit fluid to flow from the conduit 490 into a waste chamber 560, ahole 565 may comprise a capillary stop between conduits 440 and 510, ahole 570 may permit air to flow between a recess 575 (as shown in FIG.23) and a conduit 580 (as shown in FIG. 24), the hole 550 provides forair movement between the recess 545 and the conduit 440, and the hole505 permits fluid to flow from a conduit 585 (as shown in FIG. 23) tothe waste chamber 560 via optional closeable valve 590 (as shown in FIG.25). Holes 595 and 600 permit a plurality of electrodes that are housedwithin cutaways 605 and 610, respectively, to contact fluid within theconduit 490. In a specific embodiment, cutaway 610 houses a groundelectrode, and/or a counter-reference electrode, and cutaway 605 housesat least one biosensor, and optionally, a reference sensor.

Referring to FIG. 25, the conduit 440 may be configured as a sampleholding chamber that connects the sample entry port 445 to the conduit510 in the assembled cartridge 400. The cutaway 605 may house at leastone analyte biosensor, or an analyte responsive surface, together withan optional conductimetric sensor or sensors. The cutaway 610 may housea ground electrode if needed as a return current path for anelectrochemical sensor, and may also house an optional conductimetricsensor. A cutaway 615 may provide a fluid path between gasket holes 535and 540 such that fluid may pass between the conduits 490 and 510.Recess 475 houses a fluid-containing package, e.g., a rupturable pouch,in the assembled cartridge 400 that may be pierced by the spike 480because of pressure exerted upon paddle 455 upon insertion of thecartridge 400 into the reading device. Fluid from the pierced packageflows into the conduit 485. The air bladder may be comprised of therecess 465, which is sealed on its upper surface by the gasket 415. Theair bladder may be one embodiment of a pump means, and may be actuatedby pressure applied to the paddle 460, which displaces air in theconduit 580 and thereby displaces the sample from the sample chamber 440into the conduit 510.

In some embodiments, a metering means may optionally comprise the samplechamber 440 bounded by the capillary stop 565 and having along thechamber 440 length an air entry point (gasket hole 550) from thebladder. Air pressure exerted at the gasket hole 550 drives a meteredvolume of the sample past the capillary stop 565. Therefore, a meteredvolume of sample may be predetermined by a volume of the sample chamber440 between the air entry point 550 and the capillary stop 565. Anamount of the sample corresponding to this volume may be displaced intothe conduit 510 when the paddle 460 is displaced. This arrangement maytherefore provide a metering means for delivering a metered amount of anunmetered sample into the various downstream conduits of the cartridge400. The metering may be advantageous in some embodiments ifquantitation of the analyte is required. Thus, an operator may berelieved of accurately measuring the volume of the sample prior tomeasurement saving time, effort, and increasing the accuracy andreproducibility.

As shown in FIG. 26, a schematic diagram of the features of thecartridge 700 and components therein is provided. Specifically, inpreferred embodiments, the conduits and the sample chamber 705-735 maybe coated with dry reagents to amend the sample or fluid as discussedherein. The sample or fluid may be passed at least once over the dryreagent to dissolve the dry reagent. Reagents that may be used to amendsamples or fluid within the cartridge include enzymes, a water solubleprotein, a buffer, scavengers, or combinations thereof, and/or blockingagents that prevent either specific or non-specific binding reactionsamong assay compounds. A surface coating that may not be soluble buthelps prevent non-specific adsorption of assay components to the innersurfaces of the cartridge 700 may also be provided

For example, within a segment of the sample or fluid, an amendingsubstance may be preferentially dissolved and concentrated within apredetermined region of the segment. In one embodiment, this may beachieved through control of the position and movement of the segmentwithin the conduits and the sample chamber 705-735. Therefore, if only aportion of a segment, such as the leading edge, is reciprocated over theamended substance, then a high local concentration of the substance canbe achieved close to the leading edge. Alternatively, if a homogenousdistribution of the substance is desired, for example if a knownconcentration of an amending substance is required for a quantitativeanalysis, then further reciprocation of the sample or fluid may resultin mixing and an even distribution.

In preferred embodiments, a closeable valve 740 may be provided betweena first conduit and the waste chamber. In one embodiment, the valve 740may be comprised of a dried sponge material that is coated with animpermeable substance. In operation, contacting the sponge material withthe sample or a fluid may result in swelling of the sponge to fill thecavity (e.g., the valve 590 cavity as shown in FIG. 25), therebysubstantially blocking further flow of liquid into the waste chamber.Furthermore, the wetted valve 740 may also be configured to block theflow of air between the first conduit and the waste chamber, whichpermits a first pump means connected to the sample chamber to displacefluid within a second conduit, and to displace fluid from the secondconduit into the first conduit in the following manner.

After the sample is exposed to the sensor array (e.g., the biosensors)for a controlled time, the sample may be moved into a post-analyticalconduit where the sample may be amended with another reagent. The samplemay then be moved back to the sensor array and a second reaction periodmay begin. Alternately, the post-analysis conduit may serve simply toseparate the sample segment from the sensor array. Within thepost-analysis conduit may be a single closeable valve that connects anair vent of the sensor conduit to a diaphragm air pump. When the singlecloseable valve closes, the sample may be locked in the post analyticalconduit and cannot be moved back to the sensor array.

In a preferred embodiment of the present invention, the sample and afluid, e.g., a combined wash and substrate delivery fluid, may contactthe sensor array at different times during an assay sequence. The sampleand the fluid may also be independently amended with other reagents orcompounds present initially as dry coatings within respective conduitsof a test device, e.g., the cartridge. Controlled motion of the fluid bythe above-described pumping means within the cartridge further permitsmore than one substance to be amended into each fluid whenever thesample or the fluid is moved to a new region of the conduit. In thismanner, multiple amendments to each fluid may be accommodated, extendingthe complexity of automated assays that can be performed in thecartridge. Therefore, the utility of the present invention may beenhanced.

In an alternative embodiment, as shown in FIGS. 27A-27E, the cartridge900 may include a housing that comprises two complimentary halves of acartridge (e.g., the cover 901 and the base 902), which can be bondedtogether to abut and attach the two complimentary interior surfaces ofthe two halves in a closed position. In some embodiments, the cover 901and the base 902 are injection molded, for example, by machine asdisclosed in U.S. patent application Ser. No. 13/530,501, filed on Jun.22, 2012, which is incorporated herein by reference in its entirety.Preferably, the cover 901 is injection molded where a firstsubstantially rigid zone 920 is formed in a first injection molding stepand a substantially flexible zone 922 is formed in an additionalinjection molding step. Preferably, the base 902 is injection moldedwhere a second substantially rigid zone 924 is formed in a firstinjection molding step.

As shown in FIGS. 27A-27E, the substantially rigid zones 920 and 924 ofthe cover 901 and the base 902, respectively, are preferably each asingle contiguous zone; however, the molding process can provide aplurality of non-contiguous substantially rigid zones. The substantiallyflexible zone 922 is preferably a set of several non-contiguous zones.For example, the substantially flexible zone 922 around a displaceablemembrane 925 may be separate and distinct from the substantiallyflexible zone at a closeable sealing member 928. Alternatively, thesubstantially flexible zone may comprise a single contiguous zone.

In a preferred embodiment, the cartridge housing comprises a sensorrecess 930 in a portion of the substantially flexible zone. An advantageis that the sensors 935, which are disposed in the sensor recess 930preferably are made on a silicon wafer substrate, which is relativelybrittle. Thus, providing a substantially flexible sensor recess 930results in a suitable support that can protect the sensor from crackingduring assembly. Note that other non-silicon based sensors may be used,e.g., those made on a plastic substrate; however, the preferredembodiment uses sensors of the type described in U.S. Pat. Nos.5,200,051; 5,514,253; and 6,030,827, the entireties of which areincorporated herein by reference. In addition to being substantiallyflexible, sensor recess 930 may be best selected to form a liquid-tightand/or air-tight seal around the sensor perimeter, thereby ensuring thatliquids do not leak out of the conduit that covers the sensor in thefully assembled cartridge. In an alternative embodiment, sensor recess930 can be formed in a portion of the substantially rigid zone (as shownin FIG. 25) of either or both of the cover or the bottom of the housing.In this aspect, a liquid-tight and/or air-tight seal optionally may beformed by the double-sided adhesive sheet 415 or gasket (as shown inFIG. 24).

With regard to overall dimensions, the preferred embodiment of themolded parts shown in FIGS. 27A-27E include the cover 901 withdimensions of about 6.0 cm×3.0 cm×0.2 cm and the base 902 withdimensions of about 5.0 cm×3.0 cm×0.2 cm to provide a cartridge 900 withdimensions of about 6.0 cm×3.0 cm×0.4 cm. In terms of ranges, thecartridge 900 optionally has a length of from 1 to 50 cm, e.g., from 5to 15 cm, a width of from 0.5 to 15 cm, e.g., from 1 to 6 cm, and athickness of from 0.1 to 2 cm, e.g., from 0.1 to 1 cm.

Processes for Target Analyte Detection Using a Biosensor

In preferred embodiments, the invention is a process for using acartridge to determine the presence and/or concentration of a targetanalyte in a sample. The process may include introducing an unmeteredfluid sample into the sample chamber 440 of the cartridge 400 throughthe sample entry port 445 (as shown in FIGS. 22-25). Capillary stop 565prevents passage of the sample into conduit 510 at this stage, andconduit 440 is filled with the sample. Lid 435 is closed to preventleakage of the sample from the cartridge. The cartridge may then beinserted into the reading device or apparatus 387, as shown in FIG. 21and further disclosed in U.S. Pat. No. 5,821,399, which is incorporatedherein by reference in its entirety. Insertion of the cartridge into thereading apparatus activates a mechanism, which punctures thefluid-containing package located at recess 475 when the package ispressed against spike 480. Fluid is thereby expelled into the conduits485 and 490, arriving in sequence at the sensor region. The constriction520 prevents further movement of fluid because residual hydrostaticpressure is dissipated by the flow of fluid via the conduit 585 into thewaste chamber 560.

In a second step, operation of a pump means applies pressure to theair-bladder comprised of cavity 465, forcing air through the conduit 580and into conduit 440 at a predetermined location. Capillary stop 565delimits a metered portion of the original sample. While the sample iswithin sample chamber 440, it is preferably amended with a compound orcompounds (e.g., enzymes, a water soluble protein, a buffer, scavengers,or a combination thereof) present initially as a dry coating or layer(s)on the inner surface of the chamber or conduits. The metered portion ofthe sample is then expelled through the capillary stop 565 by airpressure produced within air bladder comprised of cavity 465. The samplepasses into the sensor conduit and into contact with the biosensorlocated within the cutaway 605.

To promote reaction of the analyte in the sample with the enzymeimmobilized on or near the biosensor, the sample containing the analyte(e.g., creatinine) may optionally be passed repeatedly over theelectrodes in an oscillatory motion. Preferably, the mode of operationis as described in jointly owned U.S. Pat. No. 5,096,669 which isincorporated by reference, where a calibrant fluid containing creatinineis first applied to the sensor and then the sample, e.g. blood, is movedover the sensor as the calibrant fluid is displace to a waste chamber.

Use of a cartridge with a closeable valve, preferably located betweenthe sensor chamber and the waste chamber, is herein illustrated by aspecific embodiment in which the concentration of creatinine isdetermined within a blood sample, which is introduced into the samplechamber of said cartridge. In the following time sequence, time zero(t=0) represents the time at which the cartridge is inserted into thecartridge reading device. Times are given in minutes. Between t=0 andt=1.5, the cartridge reading device makes electrical contact with theelectrodes/sensors through pads, and performs certain diagnostic tests.Insertion of the cartridge perforates the foil pouch introducingcalibrant fluid to the sensor. The diagnostic tests determine whetherfluid or sample is present in the conduits using the conductivityelectrodes; determine whether electrical short circuits are present inthe electrodes; and ensure that the biosensor and ground electrodes arethermally equilibrated to, preferably, 37° C. prior to the analytedetermination.

Various options exist for managing any temperature effect on an assay ofthis type. For example, the assay can be run in a system where thesample and other fluids and reagents are thermostated at a giventemperature, e.g., 37° C. Alternatively, the assay may be run at ambienttemperature, without any correction, or with correction to astandardized temperature based on measurement of the ambient value

A metered portion of the sample, preferably between 4 and 200 μL, morepreferably between 4 and 20 μL, and most preferably 7 μL, may be used tocontact the electrodes/sensors as described above.

While the invention has been described in terms of various preferredembodiments, those skilled in the art will recognize that variousmodifications, substitutions, omissions and changes can be made withoutdeparting from the spirit of the present invention. It is intended thatthe scope of the present invention be limited solely by the scope of thefollowing claims. In addition, it should be appreciated by those skilledin the art that a plurality of the various embodiments of the invention,as described above, may be coupled with one another and incorporatedinto a single reader device.

We claim:
 1. A method of manufacturing a biosensor comprising: formingan electrode on a wafer; microdispensing a first matrix that includescreatinine amidohydrolase (CNH), creatine amidinohydrolase (CRH), andsarcosine oxidase (SOX) to form a first layer on the electrode;microdispensing a second matrix that includes CRH to form a second layerover the first layer; and microdispensing a third matrix that includesCRH, SOX, and catalase to form a third layer over the second layer suchthat the second layer is disposed between the first layer and the thirdlayer, wherein the second layer completely covers the first layer,wherein the third layer completely covers the second layer, and whereinthe second matrix and the third matrix are different.
 2. The method ofclaim 1, wherein the first layer and the third layer are at least one ofmicrodispensed and dried at a controlled humidity in a range of about40-98% relative humidity.
 3. The method of claim 1, further comprising,prior to forming the first layer, the second layer, and the third layer,forming a silane layer on the electrode such that the silane layer isdisposed between the electrode and the first player.
 4. The method ofclaim 1, further comprising, prior to forming the first layer, thesecond layer, and the third layer, forming a gamma amino silane layer onthe electrode such that the gamma amino silane layer is disposed betweenthe electrode and the first layer.
 5. The method of claim 1, wherein adiameter of the electrode is less than a diameter of the first layer anda diameter of the second layer.
 6. The method of claim 1, wherein adiameter of the first layer is less than or equal to a diameter of thesecond layer.
 7. The method of claim 1, wherein the first matrix and thesecond third matrix are polymer matrixes.
 8. The method of claim 1,wherein the first matrix and the third matrix are selected from thegroup consisting of: polyvinyl alcohol, gelatin, acrylamide,polyethyleneglycol diacrylate, or combinations thereof.
 9. The method ofclaim 1, wherein the first matrix and the third matrix arephotoformable.
 10. The method of claim 1, wherein the first matrix andthe third matrix comprise a photoinitiator.
 11. The method of claim 10,wherein the photoinitiator is stilbazonium or dichromate.
 12. The methodof claim 1, wherein the first layer, the second layer, and the thirdlayer comprise a substantially concave shape.
 13. The method of claim 1,wherein the first layer, the second layer, and the third layer comprisea substantially convex shape.
 14. A method of manufacturing a biosensorcomprising: forming an electrode on a wafer; printing a first layer on atop surface the electrode, the first layer comprising creatinineamidohydrolase (CNH), creatine amidinohydrolase (CRH), and sarcosineoxidase (SOX); printing a second layer on a top surface of the firstlayer, the second layer comprising CRH and having a compositiondifferent from that of the first layer; and printing a third layer on atop surface of the second layer such that the second layer is disposedbetween the first layer and the third layer, the third layer comprisingCRH, SOX, and catalase and having a composition different from thesecond layer.